Although RNA interference (RNAi) technology has revolutionized the process of rapidly and efficiently generating models to study disease, it has been limited by the lack of robust control over short interfering or micro RNA (siRNA/miRNA) expression. The need for better regulation has prompted the development of novel, conditional siRNA/miRNA expression systems, all of which currently rely on the use of transcriptional controls. While these offer some advantages, it would be desirable to have siRNA expression under tighter and more versatile control. Here we propose such a system that promises exquisite regulation by integrating two dynamic and naturally occurring processes, RNAi and spliceosome-mediated RNA trans-splicing (SMaRT). SMaRT takes advantage of the native mammalian splicing machinery to reprogram the sequence of a targeted pre-mRNA through the activity of a Pre-Trans-splicing Molecule (PTM). PTMs and SMaRT have been used to correct genetic disorders in mice (e.g. hemophilia A), to image tumor cells in living animals, and to attempt suicide gene therapy of virally infected cells in culture. We propose to use SMaRT to generate novel pri-miRNAs, which will be created by the specific trans-splicing of a PTM and a unique pre- mRNA. Because the pri-miRNA is only generated upon trans-splicing to the correct pre-mRNA, these methods will prevent ectopic RNAi. To develop this SMaRT RNAi methodology we propose the following specific aims: In specific aim 1 we will develop PTMs that form novel pri-miRNAs capable of generating alpha-1 antitrypsin (AAT)-, human papillomavirus-16 (HPV16)- or TATA box binding protein (TBP)-specific miRNAs in tissue culture cells. In specific aim 2 we will develop and optimize the conditional expression of the miRNAs by altering the availability of the trans-splicing pre-mRNA. In specific aim 3 we propose to broaden the application of SMaRT RNAi by developing PTMs that upon trans-splicing to human papillomavirus-16 pre- mRNAs lead to the synthesis of miRNAs that knockdown the essential gene product TATA binding protein. This conditional silencing of TBP should result in the death of cells harboring papillomavirus genomes but not of normal cells. Thus the regulation provided by SMaRT RNAi establishes the groundwork for a new generation of conditional knockdown model systems and potential therapeutic modalities with global applications in human disease. [unreadable] [unreadable]